Glycoproteins and glycoenzymes are proteins that contain a post-translational modification wherein oligosaccharide chains (known as glycans) are covalently attached to the protein's or enzyme's polypeptide side chains. This process, which is known as glycosylation, is one of the most abundant protein post-translational modifications. It is estimated that more than half of all cellular and secretory proteins are glycosylated. (Apweiler et al., 1999, Biochim. Biophys. Acta 1473: 4-8). Although mammalian glycoprotein oligosaccharides, for example, are constructed from a limited number of monosaccharides, their structural diversity is vast due to complex branching patterns. Glycoproteins, therefore, represent a diverse group of modifications, and variants of glycoproteins or glycoenzymes (which are known as glycoforms) can impact protein or enzyme activity or function. The ability to evaluate and distinguish specific glycan structures during the preparation of recombinant enzymes can accordingly provide valuable information relating to recombinant enzyme development and further optimization of the desired glycoform content of such recombinant enzymes.
Conventional techniques which are routinely employed for glycoprotein and glycoenzyme analysis include mass spectrometry, lectin affinity chromatography and western blotting. Although these conventional methods of analysis are generally accurate, they are time consuming, require purification of the protein, and some, such as mass spectrometry, require specific expertise and are technically challenging. (Wang et al., 2006, Glycobiol. Epub.; Qiu et al., 2005, Anal. Chem. 77:2802-2809; Qiu et al., 2005, Anal Chem. 77:7225-7231; Novotny et al., 2005, J. Sep. Sci. 28:1956-1968). Accordingly, these issues make the routine use of such technologies impractical for high-throughput monitoring of enzyme glycosylation, especially during process development and manufacturing. Such technologies may also present challenges to a typical research laboratory attempting to study the impact of glycosylation on the biological properties of proteins and enzymes.
Traditionally, to provide a quantitative assessment of the glycan structure of a glycoprotein, lectin array platforms required the use of either a reliable glycoprotein-specific antibody or direct conjugation of a fluorescent dye to the glycoprotein. These antibody-based detection strategies are limited by the fact that antibody recognition of a given glycoprotein or glycoenzyme may be blocked or reduced depending on the type of glycan structure linked to the protein or enzyme, thereby allowing recognition of only a subset of the total glycoprotein pool and not the range of potential glycan structures. Antibody-based recognition may also require multiple binding and wash steps, which can add time and complexity to an analysis. While these problems can be circumvented using direct labeling of the glycoprotein, direct labeling remains limited to pure preparations of material, since the labeling techniques do not discriminate among proteins. Accordingly, direct labeling cannot be used for “dirty” or in-process samples. The utility of currently available methods for glycan analysis may be further limited because large quantities of highly purified materials may not readily be available from in-process test samples. Furthermore, purified material may only represent a subset of the initial glycoform population because the purification process is typically selective for certain glycan structures.
The identification and characterization of protein and enzyme glycoforms is essential in the development of recombinant proteins and enzymes. For example, glycosylation of recombinantly-prepared enzymes must frequently be controlled during production to maintain the efficacy and safety of such recombinant enzymes, and cell culture conditions can affect the carbohydrate structures of glycoproteins. Further understanding of cell culture conditions that can impact the carbohydrate structures of recombinantly-prepared proteins or enzymes is also important for the development of an effective and robust recombinant production process.
Improved methods and compositions are needed for the rapid, direct and systematic identification and evaluation of the glycan structures of a given protein or enzyme and their variant glycoforms. High throughput methods and compositions that are capable of efficiently assessing and distinguishing among a diverse range of glycosylation states or glycoforms, as well as determining the relative differences in the amount of glycans associated with such glycosylation states or glycoforms, would provide valuable information for drug discovery and disease therapeutics, provide valuable tools regarding ongoing research, and facilitate the optimization of recombinant production processes.